The present invention generally relates to drug testing methods for determining maternal drug use during pregnancy and fetal exposure to drugs in utero. More particularly, it relates to new and improved qualitative and quantitative methods for testing neonatal meconium tissue samples to provide unequivocal evidence of prenatal exposure to chemical agents such as drugs of abuse by identifying and quantifying the presence of these chemical agents or their known metabolites in the newborn meconium tissue samples.
There is growing concern in society today about the increasing numbers of newborn infants who are born in an undesirably unhealthy condition because they have been exposed to harmful chemical agents in utero. Perhaps the most widespread concern surrounds an apparent increase in maternal abuse of addictive drugs, both licit and illicit, during pregnancy. Primary interest has been focused on the so-called drugs of abuse, e.g., cocaine, opiates, including heroin, morphine and codeine, amphetamines, phencyclidine (PCP), and marijuana-related compounds, such as tetrahydrocannabinoids (THCs). Secondarily, other so-called legally available substances such as barbiturates, muscle relaxers, anti-anxiety agents, i.e. diazepam or lorazepam, alcohol, nicotine and other chemical agents may be shown to affect the health of a developing fetus. Although the present invention is primarily directed to drugs and their known metabolites, other chemical agents capable of becoming protein-bound may also be of interest. For example, gestational exposure to certain insecticides, herbicides, industrial chemicals, air pollutants and water pollutants may also be shown to adversely affect the exposed fetus in utero and the health of the newborn infant.
Maternal abuse, use or exposure to several known chemical agents, such as addictive drugs has been reported to lead to decreased birthsize and birthweight, decreased head circumference, decreased gestational duration and in a few cases, congenital defects. Generally, infants born to drug addicted and/or using mothers may be characterized by being compromised, in ill-health, exhibiting an early failure to thrive and possibly may be expected to show significant developmental delays or anomalies if they survive after the first few days and weeks. For example, neuronal damage may not become recognizable until later in the life of the child.
Proper medical care and treatment for the special needs of these disadvantaged individuals frequently requires that treating physicians and nurses be advised of the fact that the neonate has been exposed in utero. Moreover, the identity of which drug or chemical agent the child has been exposed to as a fetus may also be critical to early courses of treatment and ultimate survival. If latent neuronal damage was caused leading to real or apparent learning disabilities, early detection and possession of the information can really help that newborn in many ways. If learning disability problems are inevitable or likely, early extra educational efforts to assist the child may help to reduce or eliminate any adverse effects to that child. Moreover, if the child is identified early as disabled or handicapped due to in utero drug exposure, Federal educational and social assistance may be available to the child to help with the costs of raising, educating and caring for the child. The financial and human costs of delivering the high-risk neonatal medical care and continuing medical and social care and services for these infants in increasingly higher numbers is staggering.
Recent studies show that maternal abuse of addictive drugs during pregnancy is widespread and increasing. The National Association for Perinatal Addiction Research and Education estimated that approximately 11% of women studied in 36 hospitals had used illicit drugs during pregnancy. In regional areas where drug abuse is known to be a significant problem, the estimates are considerably higher. Recently, in one southern California hospital, authorities estimated that they had delivered at least about 20,000 drug addicted babies in one year. Similarly, a Chicago Municipal hospital estimated delivery of over 12,000 drug-exposed or addicted newborns per year. Conservative estimates by public health experts indicate that there are at least about 500,000 drug-exposed neonates born each year in the United States, about 10% of the annual birth rate.
The severity of this public health and societal problem has come to the attention of several legislatures. Some jurisdictions proscribe the use of addictive drugs during pregnancy and define the presence of such drugs in the neonate as a prima facie child abuse, thereby creating an immediate state interest in the care and custody of these infants. In addition to increased demands for intensive medical care and management, health care providers must also act responsibly in accordance with newly defined and increased duties imposed on them by these new statutes and regulations. Health care providers in some jurisdictions have a duty to screen and identify the drug exposed neonates and their mothers.
Earlier efforts to identify and assess those mothers who used or abused drugs during pregnancy and who thereby exposed their infant newborns to drugs in utero have included obtaining oral admissions from the mother, blood testing and urine testing. Oral histories, as has been mentioned above, are generally not forthcoming or reliable. Testing of blood tissue samples for the presence of drugs or their metabolites is generally time consuming and expensive. The recent AIDS epidemic has made collection, testing and handling of blood tissue samples unwise and undesirable. In addition, the target analytes of the drugs of abuse, typically looked for will not be present in either maternal bloodstream or the neonate's bloodstream even only six hours after the mother has taken the drug. Usually the first neonatal blood samples aren't taken until about 12-24 hours after delivery. Accordingly, blood testing of an infant newborn is expected to provide a ridiculously large number of false negative results.
Urine testing is also problematic for several reasons. Collecting adequate sample specimens from premature infants is difficult at best. Unless the fetus was exposed to the drug within one to three days immediately prior to delivery which is generally unlikely, urine screens frequently yield false negative results. Accordingly, urinalysis is generally inconclusive at best.
More recently, it has been demonstrated that meconium tissue formed in the intestines of a fetus in utero may provide the best physical evidence of maternal drug use. Meconium is a complicated heterogeneous tissue including a matrix of proteinaceous and cellular solids capable of binding, adsorbing and storing various chemical agents per se and/or their respective metabolic end products. Meconium is continuously formed and stored in the fetal intestines from late in the first trimester of gestation i.e., from between about 12-16 weeks, until birth.
Meconium forms the first several excreta of a newborn infant until a change to transitional milk stools is observed. Meconium tissue may therefore act as a biological time-capsule, in that each infant's meconium may reveal the history of the fetus in utero in terms of its exposure to various chemicals which become bound in the meconium matrix as the meconium is formed. The contents of neonatal meconium may provide evidence of exposure to drugs or other chemical agents back in time during the pregnancy, possibly as early as the later part of the first trimester and definitely from the second and third trimesters immediately before birth. The importance of the meconium tissue is that this precious sample is a one-shot deal. Unless the meconium is properly collected and analyzed a great deal of once-in-a-lifetime information is permanently lost. The personal and social implications of not having the important information the tissue can provide are very serious.
A major problem associated with collecting and studying meconium tissue is that it is extremely difficult to work with. Meconium has a thick visco-elastic nature that does not readily lend itself to typical sample analysis methods and equipment.
In U.S. Pat. No. 5,015,589 to Ostrea Jr., two distinct methods for testing a meconium sample to qualitatively determine whether or not it contains morphine and/or cocaine metabolites or cannabinoids, respectively, are described. In the assay procedure to qualitatively screen infant meconium for the presence of morphine or cocaine metabolites, a 0.5 to 1.0 gram sample of infant meconium is diluted more than 2 to 1 with an aqueous hydrochloric acid solution. The acidified and diluted sample is vortexed and poured through a glass wool filter to remove gross particulates. The filtrate is centrifuged and an aliquot portion is analyzed for morphine metabolites and cocaine metabolites by radioimmunoassay.
In the Ostrea Jr. method for qualitatively determining the presence of marijuana metabolites, a neonatal meconium sample is admixed with absolute methanol, vortexed and permitted to stand at room temperature for several minutes. Thereafter, the mixture is filtered, centrifuged and an aliquot portion of the supernate is tested for cannabinoid metabolites by radioimmunoassay.
The Ostrea Jr. methods described in U.S. Pat. No. 5,015,589 suffer from a number of shortcomings. The determinations are qualitative at best and do not offer unequivocal quantitative evidence of prenatal drug exposure needed to meet current forensic standards. Moreover, the qualitative procedures described are generally not reproducible in practice. This is believed to be primarily caused by the broad compositional variations in the heterogeneous meconium tissue in general. For example, a positive or negative result may depend on the location from which the aliquot portion of the meconium sample was taken in relation to the overall meconium specimen. This distributional variation within a single sample can often lead to false negative test results. Furthermore, the procedures are not particularly sensitive in practice. In accordance with the Ostrea Jr. method, a small sample of meconium is diluted with an aqueous acid. Ostrea Jr.'s acid reagents dilute trace amounts of target analytes found in the sample to a large degree. After the immunoassay reagents are added, the target unknowns are diluted even further, often below the minimum detectable concentrations of the immunoassay reagents themselves. The aqueous mineral acids employed are so strongly acid that they not only hydrolyze and denature the meconium matrix to release target analytes into aqueous solution, these acids may also de-nature the recognizable structure of the metabolites being assayed, further lowering their effective detectable concentrations.
Poor sensitivity and non-reproducibility experienced with the Ostrea Jr. method may also result from the fact that Ostrea Jr. requires a glass wool filtration step to remove gross particulates from the acidified mixture. It has independently been observed that untreated glass surfaces, such as those presented by the glass wool filters employed in the Ostrea Jr. method, are reactive with the sample analytes and absorb and bind target substances, thereby taking them out of the assay solutions. Untreated glass surfaces can absorb as much as 10 nanograms of drug or other metabolite for every square centimeter of untreated glass surface area that the drug containing solution is exposed to.
Another disadvantage to Ostrea Jr.'s methods is that different extraction procedures and steps must be performed on the same meconium tissue for each target analyte in order to only make what is an essentially unreliable qualitative determination for that analyte. In a commercial setting, too many separate procedures and duplicative steps disadvantageously increase the time and costs needed to implement the Ostrea Jr. method. Finally, the Ostrea Jr. patent describes making their screening determinations using radio immunoassays which are undesirable because costly special care, handling and disposal steps are required in order to use these radioactive materials.
Another prior art method for screening neonatal meconium samples is described by Rosenzweig et al. in an abstract appearing in Clinical Chemistry, Vol. 36, No. 6, (1990) at page 1023, Abstract No. 0334. In the method described in this abstract, evidence of drugs and drug metabolites may be qualitatively determined for amphetamine, cocaine metabolite, morphine metabolite and cannabinoid metabolite in neonatal meconium samples by non-radioactive immunoassay methods. In accordance with this screening method, a meconium sample is extracted with methanol, centrifuged and the supernatant is filtered through filter paper. The volume of the filtrate is reduced by evaporation and reconstituted in a phosphate buffer. The buffered sample is then analyzed by enzymatic colorimetric immunoassay or by a fluorescence polarization immunoassay method.
The non-radioactive screening methods described by Rosenzweig also have disadvantages in terms of reliability and sensitivity. The meconium methanol extract is generally too highly colored for direct reading in a colorimetric read assay. Ten fold dilutions are needed to achieve workable readability which reduces analyte concentration to at or below sensitivity limits to the assays. The method includes a paper-filter filtration step which has also been shown to act as a drug sponge undesirably absorbing target analyte out of the sample. Also, the Rosenzweig methods described in the abstract are only qualitative screens and do not provide the unequivocal quantitative evidence needed. Furthermore no special sample preparation steps are described or suggested so that the disclosed method also suffers from lot to lot variability for samples taken from the same patient sample.
In another abstract published in Clinical Chemistry, Vol. 36, No. 6 (1990) at page 1022, Abstract No. 0327, Clark et al, describe a first quantitative procedure for determining quantities of cocaine and its major metabolite, benzoylecgonine, from meconium samples. The Clark et al. method includes extracting cocaine and its metabolites from a sample of meconium with methanol and centrifuging to remove solids. The supernatant is concentrated by evaporation, reconstituted with phosphate buffer and extracted with a solid phase cation exchange column. Solid phase bound analytes are eluted into an organic solvent, derivatized and injected into a GC/MS instrument in selective ion monitoring mode. Unknown patient samples were compared with spiked negative samples and quantitative results were obtained from the standard curve. The GC/MS method was reported by Clark et al. to be able to detect cocaine or its metabolite at levels as low as 300 ng of drug per gram of meconium tested.
The earlier GC/MS confirmatory method described by Clark et al. made some progress toward providing a quantitative meconium testing method but suffers from several drawbacks. In practice, a methanol extract as described by Clark et al. results in a meconium sample fluid containing neutral fats and free fatty acids which frequently clog the solid phase extraction columns. The Clark et al. procedures also do not address variability of the meconium samples per se and do not provide for variable dispersability of the meconium in the methanol so that non-reproducible results are often obtained. The sensitivity for these confirmatory procedures was stated to be a drug concentration of about 300 ng/g of meconium for cocaine and benzoylecgonine. Nevertheless, the ability to detect even smaller amounts of drugs or their metabolites and determining exposure as early as within the second and first trimesters of pregnancy is still desired. The Clark et al. article specifically describes a quantitative GC/MS method for cocaine only with limited sensitivity. No teachings or suggestions are provided for making quantitative GC/MS assays for meconium samples for other drugs of abuse, their respective metabolites or other chemical agents of interest.
U.S. Pat. No. 5,185,267 (Ostrea) discloses another procedure for detecting maternally transferred drug metabolites in a newborn infant which is provided by weighing out a 0.2 gram portion of infant meconium and adding 5 mls of an aqueous phosphate buffer and methanol solution (&lt;30% methanol) to the sample. This is a substantial dilution of the sample with water. The sample is agitated, then centrifuged. The supernatant fluid is transferred to a microcentrifuge vessel and centrifuged again. A portion of the supernatant is assayed for drug analytes by enzyme immunoassay methods.
Accordingly, to overcome the limitations of the prior art procedures and methods, it is an object of the present invention to provide new and improved testing methods for obtaining unequivocal evidence of prenatal exposure to selected target analytes, such as the drugs of abuse and their metabolites, as well as other chemical agents.
It is another object of the present invention to provide faster, less expensive and more reliable methods for testing neonatal meconium samples to qualitatively and quantitatively determine gestational fetal exposure to not only cocaine and its metabolites, but also to opiates, amphetamines, phencyclidines and cannabinoids, as well as other licit and illicit drug analytes.
It is still another object of the present invention to provide new and improved methods for collecting and preparing neonatal meconium tissue samples for further testing to provide improved analyte separation and recovery.
It is still another object of the present invention to provide a new and improved method for making a smooth, substantially uniform time-averaged meconium tissue product for further testing to reduce the incidence of false negative results and aliquot to aliquot variability for neonatal meconium tissue specimens.
It is another object of the present invention to provide a new and improved method for liberating and extracting substantially all target analyte values of interest from a neonatal meconium sample, in relatively concentrated form in a single step, for use in a number of analytical testing procedures.
It is a further object of the present invention to provide new and improved methods meeting currently accepted forensic standards to unequivocally identify and quantitate the presence of drugs of abuse in meconium adapted for use on a commercial scale in a commercial testing laboratory.
It is still another object of the present invention to improve the quality of medical care for newborn infants in our society by providing fast, inexpensive and reliable methods for early detection of those newborns exposed to various harmful chemical agents while a fetus in utero.